Nucleotide sequences which code for the pck gene

ABSTRACT

The invention relates to isolated nucleotide sequences from Coryneform bacteria which code for the pck gene encoding the enzyme phosphoenol pyruvate carboxykinase (PEP carboxykinase). The invention also relates a process for the fermentative preparation of L-amino acids, in particular L-lysine, L-threonine, and L-glutamate by attenuation of the pck gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part of U.S. application Ser. No. 10/059,091, filed Jan. 30, 2002, which, in turn, is a Divisional of U.S. application Ser. No. 09/455,777, filed Dec. 7, 1999. U.S. application Ser. No. 09/455,777 claims priority to German application 199 50 409.1 filed on Oct. 20, 1999.

FIELD OF THE INVENTION

[0002] The invention provides nucleotide sequences from coryneform bacteria which code for the pck gene and a process for the fermentative preparation of L-amino acids, in particular L-lysine, L-threonine, and L-glutamate by attenuation of the pck gene.

DESCRIPTION OF BACKGROUND ART

[0003] Amino acids, in particular lysine and threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine.

[0004] It is known that these substances are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of its great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

[0005] Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolism products of regulatory importance and produce the desired amino acid are obtained in this manner.

[0006] Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid.

SUMMARY OF THE INVENTION

[0007] The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of

[0008] a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,

[0009] b) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for the polypeptide mentioned and is contained on the plasmid pEK-pckA (FIG. 1) or pEK-pckB (FIG. 2),

[0010] c) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,

[0011] d) polynucleotide which is complementary to the polynucleotides of a), b) or c),

[0012] the polypeptide preferably having the enzymatic activity of the phosphoenol pyruvate carboxykinase (PEP carboxykinase).

[0013] The invention also provides a preferably recombinant DNA with Corynebacterium origin which is capable of replication in coryneform microorganisms and contains at least the nucleotide sequence which codes for the pck gene, shown in SEQ ID No. 1.

[0014] The invention also provides a DNA according to claim 1 which is capable of replication, comprising

[0015] (i) the nucleotide sequence shown in SEQ ID no. 1, or

[0016] (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or

[0017] (iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and/or optionally

[0018] (iv) sense mutations of neutral function in (i) which do not modify the enzymatic activity of the protein/polypeptide.

[0019] The invention also provides

[0020] a polynucleotide according to claim 2, comprising the nucleotide sequence as shown in SEQ ID no. 1,

[0021] a polynucleotide according to claim 2, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2,

[0022] a vector containing the polynucleotide according to claim 1, in particular pEK-pckA or pEK-pckB, shown in FIGS. 1 and 2,

[0023] polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 728,

[0024] polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1103 and 2021,

[0025] polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 2022 and 3854,

[0026] polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 3885 and 3935,

[0027] and coryneform bacteria serving as the host cell, into which the Δpck deletion has been incorporated.

[0028] The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID no. 1 or a fragment thereof, and isolation of the DNA sequence mentioned.

[0029] Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.

[0030] They are also suitable for incorporation into so-called “arrays” or “DNA chips” in order to detect and determine the corresponding polynucleotides, e.g., m-RNA.

[0031] Polynucleotide sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for the phosphoenol pyruvate carboxykinase can be prepared with the polymerase chain reaction (PCR).

[0032] Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29, or 30, with a maximum length of 35 or 40, preferably at least 20, 21, 22, 23, or 24, with a maximum length of 30 or 35, very particularly at least 15, 16, 17, 18, or 19, with a maximum length of 25 or 30, successive nucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, with a maximum length of 45 or 50, or at least 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, with a maximum length of 55 or 60 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides with a maximum length of 350, 400, or 500 are optionally also suitable.

[0033] “Isolated” means separated out of its natural environment.

[0034] “Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non modified RNA or DNA or modified RNA or DNA.

[0035] The polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 2 or a fragment prepared therefrom and also to those which are at least in particular 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.

[0036] “Polypeptides” are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.

[0037] The polypeptides according to the invention include a polypeptide according to SEQ. ID No.:2, in particular those with the biological activity of phosphoenol pyruvate carboxykinase and also those which are at least 70% to 80%, preferably at least. 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID No:2 and have the activity mentioned.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be described with reference to the drawings, in which the stated base pair numbers are approximate values obtained in the context of reproducibility, and in which:

[0039]FIG. 1 is a restriction map of the plasmid pEK-pckA;

[0040]FIG. 2 is a restriction map of the plasmid pEK-pckB; and

[0041]FIG. 3 is a restriction map of the plasmid pk19mobsacBΔpck.

DETAILED DESCRIPTION

[0042] Amino acids, in particular L-lysine and L-threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine. There is therefore a general interest in providing new improved processes for the preparation of these products.

[0043] Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.

[0044] The invention also provides a process for the fermentative preparation of L-amino acids, in particular L-lysine and L-threonine, using coryneform bacteria which in particular already produce the L-amino acids and in which the nucleotide sequence(s) which code(s) for the pck gene are attenuated, in particular expressed at a low level.

[0045] The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding enzyme (protein), and optionally combining these measures.

[0046] The microorganisms which the present invention provides can produce L-amino acids, in particular lysine and threonine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among specialists for its ability to produce L-amino acids.

[0047] Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains

[0048]Corynebacterium glutamicum ATCC13032

[0049]Corynebacterium acetoglutamicum ATCC15806

[0050]Corynebacterium acetoacidophilum ATCC13870

[0051]Corynebacterium thermoaminogenes FERM BP-1539

[0052]Corynebacterium melassecola ATCC17965

[0053]Brevibacterium flavum ATCC14067

[0054]Brevibacterium lactofermentum ATCC13869 and

[0055] Brevibacterium divaricatum ATCC14020

[0056] and L-amino acid-producing mutants or strains prepared therefrom,

[0057] such as, for example, the lysine-producing strains

[0058]Corynebacterium glutamicum FERM-P 1709

[0059]Brevibacterium flavum FERM-P 1708

[0060]Brevibacterium lactofermentum FERM-P 1712

[0061]Corynebacterium glutamicum FERM-P 6463

[0062]Corynebacterium glutamicum FERM-P 6464 and

[0063] Corynebacterium glutamicum DSM5714 or

[0064] such as, for example, the L-threonine-producing strains

[0065]Corynebacterium glutamicum ATCC21649

[0066]Brevibacterium flavum BB69

[0067]Brevibacterium flavum DSM5399

[0068]Brevibacterium lactofermentum FERM-BP 269

[0069]Brevibacterium lactofermentum TBB-10

[0070]Corynebacterium glutamicum MH20-22B-DR17.

[0071] The inventors have succeeded in isolating the new pck gene of C. glutamicum which codes for the enzyme phosphoenol pyruvate carboxykinase (PEP carboxykinase) (EC 4.1.1.49).

[0072] To isolate the pck gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in E. coli. The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einführung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Börmann et al. (Molecular Microbiology 6(3), 317-326)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

[0073] To prepare a gene library of C. glutamicum in E. coli, it is also possible to use plasmids or plasmid vectors, such as, for example, pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)), pUC9 (Vieira et al., 1982, Gene, 19:259-268), pACYC177 (Chang and Cohen, Journal of Bacteriology 134, 1141-1156 (1978)) or pSC101 (Cohen and Chang, Journal of Bacteriology 132, 734-737 (1977)). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective.

[0074] The gene library is then incorporated into an indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580, 1983) or electroporation (Tauch et.al., 1994, FEMS Microbiological Letters, 123:343-347). The indicator strain is distinguished in that it has a mutation in the gene of interest which causes a detectable phenotype. In the context of the present invention, the E. coli mutant HG4 described by Goldie and Sanwal (Journal of Bacteriology 141: 1115-1121 (1980)) is of importance. This strain carried a mutation in the pck gene, as a result of which growth on succinate as the sole source of carbon is severely impaired. By transformation with a vector which contains the pck gene, growth on succinate can be re-established.

[0075] The long DNA fragments cloned with the aid of cosmids or other vectors can than be subcloned into known plasmid vectors in the form of shorter DNA fragments. Assignment of the gene according to the invention to a specific DNA section is made possible as a result. For this, plasmid vectors known from the prior art, such as e.g. pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or the pSU vectors described by Bartolomé et al. (Gene 102, 75-78 (1991)) are used. However, shuttle vectors which replicate both in Escherichia coli and in Corynebacterium glutamicum, such as e.g. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or pEK0 (Eikmanns et al., Gene 102 (1991)) are preferably used, so that investigations can be carried out in both species. Examples of these are the plasmids pEK-pckA (FIG. 1) and pEK-pckB (FIG. 2), which were prepared starting from the plasmid vector pEK0 and carry the pck gene according to the invention.

[0076] The DNA sections characterized in this manner are then again subcloned into the usual vectors suitable for DNA sequencing. Alternatively, the long DNA sections cloned in cosmids can be subcloned directly into sequencing vectors. Examples of such vectors which are suitable for DNA sequencing are the plasmids pGEM-5zf(−) or pGEM-5zf(+) from the company Promega Corporation (Promega Protocols and Application Guide, Second Edition, 1991, part number Y981, Promega Corporation, Madison, Wis., USA).

[0077] Methods of DNA sequencing are described, inter alia, by Sanger et al. (Proceedings of the National of Sciences of the United States of America USA, 74:5463-5467, 1977).

[0078] The DNA sequences obtained can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217-232(1986)), the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithm of Pearson and Lipman (Proceedings of the National Academy of Sciences U.S. Pat. No. 85,2444-2448 (1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries which exist in databanks accessible to the public. Databanks for nucleotide sequences which are accessible to the public are, for example, that of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) of that of the

[0079] National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).

[0080] The new DNA sequence of C. glutamicum which codes for the pck gene and which, as SEQ ID No. 1, is a constituent of the present invention was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the pck gene product is shown in SEQ ID No. 2. Endogenous enzymes are able to cleave the N-terminal amino acid methionine or formylmethionine of the protein.

[0081] Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention.

[0082] In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g., exchange of glycine for alanine or aspartic acid for glutamic acid in proteins are furthermore known among experts as “sense mutations” which do not lead to a fundamental change in the activity of the protein, i.e., one of neutral function. It is furthermore known that changes on the N and C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 2 are also a constituent of the invention.

[0083] In the same way, DNA sequences which hybridize with SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

[0084] Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41:255-260). The hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i.e., the polynucleotides treated with the probe, are at least 70% identical and formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0085] A b× SSC buffer at a temperature of approximately 50-60° C., for example, can be employed for the hybridization reaction. Probes can also hybridize here with polynucleotides which are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2× SSC and subsequently to 0.5× SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approximately 50-68° C. being established. It is optionally possible to lower the salt concentration to 0.1× SSC. Polynucleotide fragments which are, for example, at least 70% or at least 80% or at least 90% to 95% identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise in steps of approximately 1-2° C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g., DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

[0086] The inventors have found that coryneform bacteria produce L-amino acids, in particular lysine and threonine, in an improved manner after attenuation of the pck gene.

[0087] To achieve an attenuation, either the expression of the pck gene or the catalytic properties of the enzyme protein can be reduced or eliminated. The two measures can optionally be combined.

[0088] The reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The expert can find information on this e.g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996) and in known textbooks of genetics and molecular biology, such as e.g. the texbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genes and Clones”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

[0089] Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]”, Reports from the Julich Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Comprehensive description can be found in known textbooks of genetics and molecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).

[0090] Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e.g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).

[0091] An example of a mutated pck gene is the Δpck allele contained in the plasmid pK19mobsacBΔpck (FIG. 3). The Δpck allele contains only the 5′ and the 3′ flanks of the pck gene; a section of the coding region 1071 bp long is missing (deletion). This Δpck allele can be incorporated into coryneform bacteria by integration mutagenesis. The abovementioned plasmid pK19mobsacBΔpck, which is not capable of replication in C. glutamicum, is used for this. After transfer by conjugation or transformation and homologous recombination by means of a first “cross-over” event which effects integration and a second “cross-over” event which effects excision in the pck gene, the incorporation of the Δpck allele is achieved and a total loss of the enzyme function in the particular strain is achieved.

[0092] Instructions and explanations on integration mutagenesis are to be found, for example, in Schwarzer and Pühler (Bio/Technology 9,84-87 (1991)) or Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)).

[0093] Examples of amino acid-producing strains of coryneforme bacteria with an attenuated pck gene are the lysine-producing strain MH20-22BΔpck and the threonine-producing strain DM368-2Δpck.

[0094] In addition, it may be advantageous for the production of L-amino acids to over-express one or more enzymes of the particular biosynthesis route, in addition to attenuation of the pck gene.

[0095] Thus, for example, for the preparation of L-lysine

[0096] at the same time the dapA gene which codes for dihydrodipicolinate synthase can be over-expressed (EP-B 0 197 335), or

[0097] at the same time a DNA fragment which imparts S-(2-aminoethyl)-cysteine resistance can be amplified (EP-A 0 088 166).

[0098] Thus, for example, for the preparation of L-threonine

[0099] at the same time the hom gene which codes for homoserine dehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72 (1988)) or the hom^(dr) or hom_(FBR) allele which codes for a “feed back resistant” homoserine dehydrogenase (Archer et al., Gene 107, 53-59 (1991); Reinscheid et al., Journal of Bacteriology 173, 3228-3230 (1991)) can be over-expressed.

[0100] In addition to attenuation of the pck gene it may furthermore be advantageous, for the production of L-amino acids, in particular lysine and threonine, to eliminate undesirable side reactions (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0101] The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids, in particular L-lysine and L-threonine. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0102] The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture. Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during culturing in a suitable manner.

[0103] Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing is continued until a maximum of the desired L-amino acid has formed. This target is usually reached within 10 hours to 160 hours.

[0104] The following microorganism has been deposited at the Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

[0105]Escherichia coli strain DH5α/pK19mobsacBΔpck as DSM 13047

[0106] The process according to the invention is used for the fermentative preparation of L-amino acids, in particular L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine, L-glutamate, and L-methionine, with coryneform bacteria, in particular the preparation of L-lysine, L-threonine, and L-glutamate.

EXAMPLES

[0107] The present invention is explained in more detail in the following with the aid of the following examples.

[0108] For this purpose, inter alia, experiments were carried out with the lysine producer Corynebacterium glutamicum strain MH20-22B and the threonine producer Brevibacterium flavum strain DM368-2. Strain MH20-22B has been deposited as DSM5715 (EP-B-0435 132) and strain DM368-2 as DSM5399 EP-B-0385 940) at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

Example 1 Isolation of the pck Gene

[0109] To isolate the PEP carboxykinase gene (pck) from C. glutamicum a cosmid gene library was set up on the basis of the cosmid pHC79 (Hohn and Collins, Gene 11 (1980) 291-298) by a known method (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). For this, chromosomal DNA was isolated from C. glutamicum ATCC13032 (Eikmanns et al., Microbiology 140 (1994) 1817-1828) and partly digested with the restriction enzyme Sau3A. After ligation of the resulting fragments into the BamHI cleavage site of the cosmid pHC79, the batch was packed into the protein envelope of the lambda bacteriophage and the E. coli strain ED8654 (Murray et al. Molecular and General Genetics 150 (1997) 53-61) was transfected with this. The recombinant cosmids were packed into the protein envelope of the lambda phage by a method of Sternberg et al. (Gene 1 (1979) 255-280), and the transfection of E. coli ED8654 was carried out by a method of Sambrook et al. (Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). The corresponding cosmids were isolated from a total of 30 resulting recombinant E. coli clones (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and subjected to a restriction analysis with the enzyme HindIII. It was found that 24 of the cosmids possessed inserts, and that the inserts had sizes of approximately 35 kb. A total of 2200 cosmid-carrying E. coli clones were combined, and the cosmid DNA was prepared from this mixture by known processes (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press).

[0110] To isolate the pck gene from C. glutamicum, the cosmide gene library was transformed into the PEP carboxykinase-defective E. coli mutant HG4 (Goldie and Sanwal, Journal of Bacteriology 141 (1980) 115-1121) by known processes (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). Because it is PEP carboxykinase-defective, the mutant HG4 is no longer capable of growing on succinate as the only source of carbon. After transformation of the cosmid gene library into this mutant, a total of 1200 clones were obtained. Of these, a total of two clones showed growth on M9 minimal medium (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) with succinate (0.4%) as the only source of carbon. After isolation of the corresponding cosmids (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) from these clones and renewed transformation into the E. coli mutant HG4, the resulting clones were again capable of growing on M9 medium with succinate as the only source of carbon.

[0111] To limit the pck gene from C. glutamicum to a smaller fragment, the two complementing cosmids were digested with the restriction enzymes XhoI, ScaI and PvuII and separated in an electrical field on a 0.8% agarose gel by known methods (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press). Fragments in the size range above 3.0 kb were isolated from the gel by electroelution (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and ligated into the SalI (XhoI-digestion) or into the Klenow-treated EcoRI cleavage site (ScaI and PvuII digestion) of the vector pEK0 (Eikmanns et al., Gene 102 (1991) 93-98). E. coli HG4 was transformed with the ligation batches and the resulting transformants were investigated again for their ability to grow on succinate as the sole source of carbon. Seven clones with plasmids which allowed growth of the mutant HG4 on succinate were found in the transformation batch with the PvuII ligation batch. The corresponding plasmids were isolated from the recombinant strains and subjected to restriction mapping. It was found that all seven plasmids carried the same 4.3 kb PvuII insert, three in one orientation and four in the other. The newly constructed plasmids were called pEK-pckA and pEK-pckB, depending on the orientation of the insert in the vector. The restriction maps of the two plasmids are shown in FIGS. 1 and 2.

Example 2 Sequencing of the pck Structural Gene and Adjacent Regions

[0112] For the sequencing, the approximately 3.9 kb EcoRI fragment was isolated from pEK-pckA (an EcoRI cleavage site here originates from the vector pEK0) by known methods. The overhanging ends of the fragment were filled up to blunt ends with Klenow polymerase (Sambrook et al., Molecular Cloning, A Laboratory Handbook, 1989, Cold Spring Harbor Laboratory Press) and ligated into the EcoRV cleavage site of the vector pGEM-5Zf(+)(Promega Corporation, Madison, Wis., USA). The insertion of the plasmid thus generated was sequenced by the chain-stopping sequencing method (Sanger et al., Proceedings of the National Academy of Sciences USA, 74 (1977) 5463-5467). It is shown as SEQ ID No. 1. The resulting nucleotide sequence of 3935 bp was analysed with the HUSAR program package (release 3.0) of the Deutsche Krebsforschungszentrum (DKFZ=German Cancer Research Centre, Heidelberg, Germany). The sequence analysis of the fragments showed an open reading frame of 1830 bp in length, which codes for a protein consisting of 610 amino acids.

Example 3 Over-Expression of the pck Gene

[0113] The plasmids pEK-pckA and pEK-pckB were introduced into the C. glutamicum strain ATCC13032 by electroporation with subsequent selection on kanamycin(50 μg/ml)-containing BHI agar plates (Liebl et al., FEMS Microbiology Letters 65 (1989) 299-304) and the resulting strains were called ATCC13032/pEK-pckA and ATCC13032/pEK-pckB. These two strains and the starting strain were cultured in Luria-Bertani complex medium [Sambrook et al., Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press] and the PEP carboxykinase test was carried out in accordance with the method described by Bentle and Lardy [Journal of Biological Chemistry 251 (1976) 2916-2921]. The result of the analysis is shown in table 1, and shows that the PEP carboxykinase activity in the two strains with the plasmids pEK-pckA and pEK-pckB is 10 to 12 times higher than in the starting strain. TABLE 1 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) ATCC13032  120 ATCC13032/pEK-pckA 1270 ATCC13032/pEK-pckB 1510

Example 4 Preparation of an Integration Plasmid for Deletion Mutagenesis of the pck Gene

[0114] For inactivation of the PEP carboxykinase gene, the EcoRI-SacI fragment of the pck gene was isolated from the vector pEK-pckB (FIG. 2) and ligated into the vector pGEM-7Zf(+) (Promega Corporation, Madison, Wis., USA). A pck-internal 1.07 kb HindII-HindIII fragment was deleted from the resulting plasmid, and the pck gene with the 1.07 kb deletion was then isolated as the BfrI-SacI frament and, after the overhanging ends had been filled up, was ligated into the vector pk19mobsacB, which is non-replicative in C. glutamicum (Schäfer et al., Gene 145, 69-73 (1994)). In the integration plasmid pK19mobsacBΔpck (FIG. 3) constructed in this way, the 5′ region of the pck gene (350 bp) is directly adjacent to the 3′ region of the pck gene (340 bp); in the genome, the two regions are separated from one another by 1071 bp. Up to this step, all clonings were carried out in E. coli DH5α as the host.

Example 5 Deletion Mutagenesis of the pck Gene in the Lysine Producer MH20-22B

[0115]E. coli S17-1 was then transformed with the integration plasmid pK19mobsacBΔpck (Simon et al., Bio/Technology 1,784-791 (1983)). This strain allows transfer of a plasmid to Corynebacterium glutamicum by conjugation (Schäfer et al., Journal of Bacteriology 172 (1990) 1663-1666). The lysine production strain C. glutamicum MH20-22B was used as the recipient of the conjugation (Schrumpf et al., Applied Microbiology and Biotechnology 37 (1992) 566-571)). Several transconjugants were obtained from the conjugation between E. coli S17-1/pk19mobsacBΔpck and C. glutamicum MH20-22B and subsequent selection on Luria-Bertani agar plates with kanamycin (25 μg/ml) and nalidixic acid (50 μg/ml). For selection for the second recombination event, which is to lead to excision of the vector together with the pck gene, these transconjugants were cultured on antibiotic-free Luria-Bertani complex medium [Sambrook et al; Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press] with 1% glucose and then plated out on the same medium plus 10% sucrose. The sacB gene present on the vector pk19mobsacB codes for the enzyme levan sucrase and leads to synthesis of levan from sucrose. Since levan is toxic to C. glutamicum, only C. glutamicum cells which have lost the integration plasmid can grow on sucrose-containing medium (Jäger et al., Journal of Bacteriology 174 (1992) 5462-5466). 30 sucrose-resistant clones were investigated for their kanamycin sensitivity. For 11 of the clones tested, in addition to the sucrose resistance, the desired kanamycin sensitivity could also be confirmed. In these 11 clones, the vector background had therefore been excised again. Whether the desired deletion had also taken place was tested by analysis by means of the polymerase chain reaction (PCR). For this, chromosomal DNA was liberated from a colony of the starting strain and from colonies of the 11 kanamycin-sensitive clones. For this, the particular colony was removed from the agar plate with a toothpick, suspended in 50 μl H₂O and incubated for 5 minutes at 95° C. 1 μl portions of the resulting solution were each employed as templates in the PCR. Oligonucleotides which cover the regions from nucleotide 2136 to 2158 and from 3815 to 3793 in SEQ ID No. 1 were used as primers. The PCR conditions were: prior denaturing: 150 seconds at 94° C.; denaturing 60 seconds at 94° C.; hybridization 30 seconds at 60° C.; amplification 120 seconds at 72° C.; 30 cycles, end extension 240 seconds at 72° C. On the basis of the primers chosen, a PCR product of 1.68 kb was expected in the batch with the DNA of the starting strain. A PCR product of 0.61 kb was expected in the PCR with the pck deletion mutant. A PCR product 0.61 kb in size was obtained with one clone. The desired deletion of the internal 1071 bp pck fragment in this clone was thereby demonstrated. The clone was called MH20-22BΔpck. The 1.68 kb PCR product was detected in the batches of the other clones. In these, the vector had thus been excised such that the genomic starting situation was re-established.

[0116] The strain MH20-22BΔpck and the starting strain MH20-22B were grown in Luria-Bertani complex medium plus 1% glucose and the PEP carboxykinase test was carried out in accordance with the method described by Bentle and Lardy (Journal of Biological Chemistry 251 (1976) 2916-2921). The result of the analysis (table 2) shows that PEP carboxykinase activity is no longer detectable in the mutant MH20-22BΔpck, in contrast to the starting strain MH20-22B. TABLE 2 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) MH20-22B 65 MH20-22BΔpck  <3*

Example 6 Production of L-lysine

[0117] To investigate the effect of the inactivation of the PEP carboxykinase gene on lysine production, the strain MH20-22B (Schrumpf et al., Applied Microbiology and Biotechnology 1992, 37:566-571) and the PEP carboxykinase-negative mutant MH20-22BΔpck (example 5) were cultured in Luria-Bertani complex medium plus 1% glucose and the fermentation medium CGXII (Keilhauer et al., Journal of Bacteriology 1993, 175:5595-5603) was inoculated from the two pre-cultures (5% inokulum, optical density at 600 nm approximately 0.5). The medium additionally comprised 3 mM leucine, since the two strains are leucine-auxotrophic. The batches each comprise 60 ml culture, obtained in 500 ml conical flasks with baffles. After culture for 24 hours at 28° C. on a rotary shaking machine of the Certomat S/50 type (B. Braun Biotech International, Melsungen, Germany) at 120 rpm, the concentration of the lysine secreted into the medium was determined.

[0118] The amino acid concentration was determined by means of high pressure liquid chromatography (Jones und Gilligan, Journal of Chromatography 1983, 266:471-482). The result of the fermentation is shown in table 3. TABLE 3 Lysine concentration in the culture supernatant of the strains MH20-22B and MH20-22BΔpck Strain L-Lysine (mM) MH20-22B 54 MH20-22BΔpck 65

Example 7 Deletion Mutagenesis of the pck Gene in the Threonine Producer DM368-2

[0119] As in the case of the lysine producer MH20-22B, using the E. coli strain S17-1/pk19mobsacBΔpck a conjugation with the threonine producer DM368-2 with subsequent selection for the first and second recombination was carried out (see example 5). Of 30 sucrose-resistant clones, 14 were kanamycin-sensitive. Of these, the 1071 bp deletion in the pck gene could be detected with the aid of the PCR analysis described in example 5 in two clones, called DM368-2Δpck16 and DM368-2Δpck18.

[0120] An enzyme test with the starting strain DM368-2 and the two pck deletion strains DM368-2Δpck16 and DM368-2Δpck18, carried out as described in example 5, showed that no PEP carboxykinase activity is detectable in these mutants (Table 4). TABLE 4 PEP carboxykinase activity in various strains PEP carboxykinase Strain (nmol min⁻¹ mg protein⁻¹) DM368-2 79 DM368-2BΔpck16  <3* DM368-2BΔpck18  <3*

Example 8 Production of L-threonine

[0121] Analogously to the experiments on L-lysine production, the accumulation of threonine in the culture supernatant of the PEP carboxykinase-defective strain DM368-2BΔpck16 was investigated in comparison with the starting strain DM368-2. For this, the two strains were cultured in Luria-Bertani complex medium plus 1% glucose and the fermentation medium CGXII was inoculated from the precultures. After culture for 24 hours at 28° C. on a rotary shaking machine at 120 rpm, the concentration of the threonine secreted into the medium was determined.

[0122] The amino acid concentration was determined by means of high pressure liquid chromatography (see above). The result of the fermentation is shown in table 5. TABLE 5 Threonine concentration in the culture supernatant of the strains DM 368-2 and DM 368-2Δpck16 Strain L-Threonine (mM) DM368-2  8 DM368-2Δpck16 22

Example 9 Deletion Mutagenesis of the pck Gene in C. glutamicum Wild-Type Strain ATCC13032

[0123] A conjugation with the C. gutamicum wild type-strain ATCC13032 was carried out with the E. coli strain S 17-1/pK19 mobsacBΔpck (example 4) as in the case of the lysine producer MH20-22B (example 5) and of the threonine producer DM368-2 (example 7). The selection on the first and second recombination event was carried out as in example 5. Of 52 saccharose-resistant clones, 8 were kanamycin-sensitive. Of these, the 1071 bp deletion in the pck gene was able to be demonstrated with the aid of the FCR analysis described in example 5 in three clones designated as ATCC13032Δpck2, ATCC13032Δpck29 and ATCC13032Δpck30.

[0124] An enzyme test with the initial strain ATCC13032 and two of the three pck deletion strains, ATCC13032Δpck2 and ATCC13032Δpck29, carried out as described as in example 5, showed that no PEP carboxkinase activity can be demonstrated in both mutants (table 6). TABLE 6 PEP carboxykinase activity In different strains Strain PEP carboxykinase ATCC13032 132  ATCC13032Δpck2 <3* ATCC13032Δpck29 <3*

Example 10 Production of L-glutamate

[0125] The accumulation of L-glutamate in the culture supernatant of the PEP carboxykinase-defective strains ATCC13032Δpck2 and ATCC13032Δpck29 in comparison to the initial strain ATCC13032 was also investigated in analogy with the experiments on the production of L-lysine and of L-threoninc (examples 6, 8). To this end the three strains were cultivated in Luria-Bertani complex [complexing] medium plus 1% glucose and the fermentation medium CGXII (see example 6) from these precultures inoculated. 15 mg Tween 60 per ml were added to the medium 4 hours after the inoculation for the induction of the production of L-glutamate. After cultivation for 24 hours at 30° C. on a rotary agitator at 120 rmp the concentration of the glutamate excreted into the medium was determined.

[0126] The determination of the concentration of amino acid took place by means of high-pressure liquid chromatography (see example 6).

[0127] The result of the fermentation is shown in table 7, in which the indicated values represent average values from two experiments in each instance. It turns out that the inactivation of the PEP carboxykinase gene results in a 4 to 5-fold increase of the concentration of glutamate in the medium. TABLE 7 Concentration of glutamate in the culture supernatant of the strains ATCC13032, ATCC13032Δpck2 and ATCC13032Δpck29 Strain L-glutamate (mM) ATCC13032 11.24 ATCC13032Δpck2 49.45 ATCC13032Δpck29 52.17

[0128] In the Figures, the abbreviations and designations that are used have the following meanings: sacB: sacB gene ori V: Replication origin V ori T: Replication origin for the transfer Km-r: Kanamycin resistance KpnI: Cleavage site of the restriction enzyme KpnI HindIII: Cleavage site of the restriction enzyme HindIII HindII: Cleavage site of the restriction enzyme HindII PstI: Cleavage site of the restriction enzyme PstI SphI: Cleavage site of the restriction enzyme SphI XbaI: Cleavage site of the restriction enzyme XbaI SalI: Cleavage site of the restriction enzyme SalI SacI: Cleavage site of the restriction enzyme SacI BfrI: Cleavage site of the restriction enzyme BfrI ScaI: Cleavage site of the restriction enzyme ScaI BamHI: Cleavage site of the restriction enzyme BamHI EcoRI: Cleavage site of the restriction enzyme EcoRI pck′: 3′ terminal fragment of the pck gene pck″: 5′ terminal fragment of the pck gene pck: pck gene

[0129]

1 2 1 3935 DNA Corynebacterium glutamicum CDS (2022)..(3851) 1 ctggcagttc tcctaattga tcgcgggaat tatcagaaat agacattatt tgttattttt 60 cctgttcaac tttaaaactt caatattcgt gagtttggat gaatccctag agcactacct 120 tttagacctc tcgctgcaat ttaggccagt tgagatttaa gctttccgac gattcttctc 180 attactgcaa tcgtaccggc gatggtggac acgatgacat gaaagagcat taaagcaatc 240 aagtacaggc tgaagtagtt aaaccactcc actccggtgc tctgtgataa aaaatgcgca 300 cccaaactca aagtgccaac tgggaaggta ctggcccacc atgtggggct gtatgtcgcc 360 cctttgaaaa cagctctgta gaacacaaag tgagcgatgg ctcccagagg aatcgtaaaa 420 attcccatga tgatgccgta aataatgccc attgtgattg ctgtcttgga tccaaaggac 480 gcaccgatga gctgagctgc tgcagtggat tggcccacca tacccaaagg aatccatgat 540 gttggtgttg ccatcagtgg gatgccctgc gccttggggc cgaaatagta gaaatacact 600 cgggtaaaaa ctgctggtgc agacgccaaa gttaaaagga agagcccgaa agaaacccac 660 agcatcgccg gaagttcaaa gtgctcatgg agttgtgctg ccgaggtgga agcaaccatc 720 ggcgtgacaa gaggaagacc ccacgcaaaa gttggtgtgc ccgccttaga tcgcaaaatg 780 gccgttatat ataaggaata ggcaacaagt cccacggctg tgccaataga ccagcacaca 840 aacataaatc cccacagatc atcacccaaa actacggggc ttgcagttcc caatgcgatc 900 aaacccatgg acagcattgc ccatgccggc atgacttcag ttttgaatga aggagagcgg 960 tagattagcc aaccgccaat aatgacaatt gccaccacaa cagctaacgc gaagaagaaa 1020 tctgcgacga ctggaaaacc atggattttc aacagtgatg acaacaatga gatgcccatg 1080 agggaaccag cccacgaggg gccaggtgga ggtaagaccg cagcgtagct tttggtcgaa 1140 gaaggagtgg gcatgcccat tactttaagc ctttggggca gtgaaaccgc taaatgggag 1200 cgttgtgcgc tcgatcactg gtctagacct ttgggctcca aaagttgcaa tttcgcgaat 1260 acttcaacac ttgtttgcaa tgtttgttaa taaatgggtt cgctagtgga ttctgtcgtt 1320 agtactggcc gtcgtggtgg ggtcatgtat ttaggtaggg caaagttaag atcagagcac 1380 tttttgatac gactaactgg atataacctt tggggtaacg tggggatgtg tgtgagtaat 1440 tttcaaagta tttaaaaggg ggatctaggg taaaaatttg gcttcaagta catatcttta 1500 gttcggtagt tgagggcggg tggtgacagt gcgggcatgc atgtgagtgt aaatgttgtt 1560 ttaaaaaggt gtgtactgac agtgggccgg tttgtgctgg tcggccacta gcggagtgct 1620 tggattgtga tggcagggta agggaaaggg attaccatta ccgctgttct tggcgttttg 1680 ttgcctattg tccgaatgtt aagtgttaat ggtgggaaaa ctgggaaagt tgtcccctgg 1740 aatgtgtgag aattgcccaa atctgaaccc aatggccatg gacggggaat gaactgtcgg 1800 agaacggttg aggttaattc ttgaaaccac ccccaaaata ggctatttaa acgggtgctc 1860 tcatattaaa gaaagtgtgt agatgcgtgt gggcaggggg taggtccact ggtaatgaca 1920 aatgtgtccg ttgtctcacc taaagtttta actagttctg tatctgaaag ctacgctagg 1980 gggcgagaac tctgtcgaat gacacaaaat ctggagaagt a atg act act gct gca 2036 Met Thr Thr Ala Ala 1 5 atc agg ggc ctt cag ggc gag gcg ccg acc aag aat aag gaa ctg ctg 2084 Ile Arg Gly Leu Gln Gly Glu Ala Pro Thr Lys Asn Lys Glu Leu Leu 10 15 20 aac tgg atc gca gac gcc gtc gag ctc ttc cag cct gag gct gtt gtg 2132 Asn Trp Ile Ala Asp Ala Val Glu Leu Phe Gln Pro Glu Ala Val Val 25 30 35 ttc gtt gat gga tcc cag gct gag tgg gat cgc atg gcg gag gat ctt 2180 Phe Val Asp Gly Ser Gln Ala Glu Trp Asp Arg Met Ala Glu Asp Leu 40 45 50 gtt gaa gcc ggt acc ctc atc aag ctc aac gag gaa aag cgt ccg aac 2228 Val Glu Ala Gly Thr Leu Ile Lys Leu Asn Glu Glu Lys Arg Pro Asn 55 60 65 agc tac cta gct cgt tcc aac cca tct gac gtt gcg cgc gtt gag tcc 2276 Ser Tyr Leu Ala Arg Ser Asn Pro Ser Asp Val Ala Arg Val Glu Ser 70 75 80 85 cgc acc ttc atc tgc tcc gag aag gaa gaa gat gct ggc cca acc aac 2324 Arg Thr Phe Ile Cys Ser Glu Lys Glu Glu Asp Ala Gly Pro Thr Asn 90 95 100 aac tgg gct cca cca cag gca atg aag gac gaa atg tcc aag cat tac 2372 Asn Trp Ala Pro Pro Gln Ala Met Lys Asp Glu Met Ser Lys His Tyr 105 110 115 gct ggt tcc atg aag ggg cgc acc atg tac gtc gtg cct ttc tgc atg 2420 Ala Gly Ser Met Lys Gly Arg Thr Met Tyr Val Val Pro Phe Cys Met 120 125 130 ggt cca atc agc gat ccg gac cct aag ctt ggt gtg cag ctc act gac 2468 Gly Pro Ile Ser Asp Pro Asp Pro Lys Leu Gly Val Gln Leu Thr Asp 135 140 145 tcc gag tac gtt gtc atg tcc atg cgc atc atg acc cgc atg ggt att 2516 Ser Glu Tyr Val Val Met Ser Met Arg Ile Met Thr Arg Met Gly Ile 150 155 160 165 gaa gcg ctg gac aag atc ggc gcg aac ggc agc ttc gtc agg tgc ctc 2564 Glu Ala Leu Asp Lys Ile Gly Ala Asn Gly Ser Phe Val Arg Cys Leu 170 175 180 cac tcc gtt ggt gct cct ttg gag cca ggc cag gaa gac gtt gca tgg 2612 His Ser Val Gly Ala Pro Leu Glu Pro Gly Gln Glu Asp Val Ala Trp 185 190 195 cct tgc aac gac acc aag tac atc acc cag ttc cca gag acc aag gaa 2660 Pro Cys Asn Asp Thr Lys Tyr Ile Thr Gln Phe Pro Glu Thr Lys Glu 200 205 210 att tgg tcc tac ggt tcc ggc tac ggc gga aac gca atc ctg gca aag 2708 Ile Trp Ser Tyr Gly Ser Gly Tyr Gly Gly Asn Ala Ile Leu Ala Lys 215 220 225 aag tgc tac gca ctg cgt atc gca tct gtc atg gct cgc gaa gaa gga 2756 Lys Cys Tyr Ala Leu Arg Ile Ala Ser Val Met Ala Arg Glu Glu Gly 230 235 240 245 tgg atg gct gag cac atg ctc atc ctg aag ctg atc aac cca gag ggc 2804 Trp Met Ala Glu His Met Leu Ile Leu Lys Leu Ile Asn Pro Glu Gly 250 255 260 aag gcg tac cac atc gca gca gca ttc cca tct gct tgt ggc aag acc 2852 Lys Ala Tyr His Ile Ala Ala Ala Phe Pro Ser Ala Cys Gly Lys Thr 265 270 275 aac ctc gcc atg atc act cca acc atc cca ggc tgg acc gct cag gtt 2900 Asn Leu Ala Met Ile Thr Pro Thr Ile Pro Gly Trp Thr Ala Gln Val 280 285 290 gtt ggc gac gac atc gct tgg ctg aag ctg cgc gag gac ggc ctc tac 2948 Val Gly Asp Asp Ile Ala Trp Leu Lys Leu Arg Glu Asp Gly Leu Tyr 295 300 305 gca gtt aac cca gaa aat ggt ttc ttc ggt gtt gct cca ggc acc aac 2996 Ala Val Asn Pro Glu Asn Gly Phe Phe Gly Val Ala Pro Gly Thr Asn 310 315 320 325 tac gca tcc aac cca atc gcg atg aag acc atg gaa cca ggc aac acc 3044 Tyr Ala Ser Asn Pro Ile Ala Met Lys Thr Met Glu Pro Gly Asn Thr 330 335 340 ctg ttc acc aac gtg gca ctc acc gac gac ggc gac atc tgg tgg gaa 3092 Leu Phe Thr Asn Val Ala Leu Thr Asp Asp Gly Asp Ile Trp Trp Glu 345 350 355 ggc atg gac ggc gac gcc cca gct cac ctc att gac tgg atg ggc aac 3140 Gly Met Asp Gly Asp Ala Pro Ala His Leu Ile Asp Trp Met Gly Asn 360 365 370 gac tgg acc cca gag tcc gac gaa aac gct gct cac cct aac tcc cgt 3188 Asp Trp Thr Pro Glu Ser Asp Glu Asn Ala Ala His Pro Asn Ser Arg 375 380 385 tac tgc gta gca atc gac cag tcc cca gca gca gca cct gag ttc aac 3236 Tyr Cys Val Ala Ile Asp Gln Ser Pro Ala Ala Ala Pro Glu Phe Asn 390 395 400 405 gac tgg gaa ggc gtc aag atc gac gca atc ctc ttc ggt gga cgt cgc 3284 Asp Trp Glu Gly Val Lys Ile Asp Ala Ile Leu Phe Gly Gly Arg Arg 410 415 420 gca gac acc gtc cca ctg gtt acc cag acc tac gac tgg gag cac ggc 3332 Ala Asp Thr Val Pro Leu Val Thr Gln Thr Tyr Asp Trp Glu His Gly 425 430 435 acc atg gtt ggt gca ctg ctc gca tcc ggt cag acc gca gct tcc gca 3380 Thr Met Val Gly Ala Leu Leu Ala Ser Gly Gln Thr Ala Ala Ser Ala 440 445 450 gaa gca aag gtc ggc aca ctc cgc cac gac cca atg gca atg ctc cca 3428 Glu Ala Lys Val Gly Thr Leu Arg His Asp Pro Met Ala Met Leu Pro 455 460 465 ttc att ggc tac aac gct ggt gaa tac ctg cag aac tgg att gac atg 3476 Phe Ile Gly Tyr Asn Ala Gly Glu Tyr Leu Gln Asn Trp Ile Asp Met 470 475 480 485 ggt aac aag ggt ggc gac aag atg cca tcc atc ttc ctg gtc aac tgg 3524 Gly Asn Lys Gly Gly Asp Lys Met Pro Ser Ile Phe Leu Val Asn Trp 490 495 500 ttc cgc cgt ggc gaa gat gga cgc ttc ctg tgg cct ggc ttc ggc gac 3572 Phe Arg Arg Gly Glu Asp Gly Arg Phe Leu Trp Pro Gly Phe Gly Asp 505 510 515 aac tct cgc gtt ctg aag tgg gtc atc gac cgc atc gaa ggc cac gtt 3620 Asn Ser Arg Val Leu Lys Trp Val Ile Asp Arg Ile Glu Gly His Val 520 525 530 ggc gca gac gag acc gtt gtt gga cac acc gct aag gcc gaa gac ctc 3668 Gly Ala Asp Glu Thr Val Val Gly His Thr Ala Lys Ala Glu Asp Leu 535 540 545 gac ctc gac ggc ctc gac acc cca att gag gat gtc aag gaa gca ctg 3716 Asp Leu Asp Gly Leu Asp Thr Pro Ile Glu Asp Val Lys Glu Ala Leu 550 555 560 565 acc gct cct gca gag cag tgg gca aac gac gtt gaa gac aac gcc gag 3764 Thr Ala Pro Ala Glu Gln Trp Ala Asn Asp Val Glu Asp Asn Ala Glu 570 575 580 tac ctc act ttc ctc gga cca cgt gtt cct gca gag gtt cac agc cag 3812 Tyr Leu Thr Phe Leu Gly Pro Arg Val Pro Ala Glu Val His Ser Gln 585 590 595 ttc gat gct ctg aag gcc cgc att tca gca gct cac gct taaagttcac 3861 Phe Asp Ala Leu Lys Ala Arg Ile Ser Ala Ala His Ala 600 605 610 gcttaagaac tgctaaataa caagaaaggc tcccaccgaa agtgggagcc tttcttgtcg 3921 ttaagcgatg aatt 3935 2 610 PRT Corynebacterium glutamicum 2 Met Thr Thr Ala Ala Ile Arg Gly Leu Gln Gly Glu Ala Pro Thr Lys 1 5 10 15 Asn Lys Glu Leu Leu Asn Trp Ile Ala Asp Ala Val Glu Leu Phe Gln 20 25 30 Pro Glu Ala Val Val Phe Val Asp Gly Ser Gln Ala Glu Trp Asp Arg 35 40 45 Met Ala Glu Asp Leu Val Glu Ala Gly Thr Leu Ile Lys Leu Asn Glu 50 55 60 Glu Lys Arg Pro Asn Ser Tyr Leu Ala Arg Ser Asn Pro Ser Asp Val 65 70 75 80 Ala Arg Val Glu Ser Arg Thr Phe Ile Cys Ser Glu Lys Glu Glu Asp 85 90 95 Ala Gly Pro Thr Asn Asn Trp Ala Pro Pro Gln Ala Met Lys Asp Glu 100 105 110 Met Ser Lys His Tyr Ala Gly Ser Met Lys Gly Arg Thr Met Tyr Val 115 120 125 Val Pro Phe Cys Met Gly Pro Ile Ser Asp Pro Asp Pro Lys Leu Gly 130 135 140 Val Gln Leu Thr Asp Ser Glu Tyr Val Val Met Ser Met Arg Ile Met 145 150 155 160 Thr Arg Met Gly Ile Glu Ala Leu Asp Lys Ile Gly Ala Asn Gly Ser 165 170 175 Phe Val Arg Cys Leu His Ser Val Gly Ala Pro Leu Glu Pro Gly Gln 180 185 190 Glu Asp Val Ala Trp Pro Cys Asn Asp Thr Lys Tyr Ile Thr Gln Phe 195 200 205 Pro Glu Thr Lys Glu Ile Trp Ser Tyr Gly Ser Gly Tyr Gly Gly Asn 210 215 220 Ala Ile Leu Ala Lys Lys Cys Tyr Ala Leu Arg Ile Ala Ser Val Met 225 230 235 240 Ala Arg Glu Glu Gly Trp Met Ala Glu His Met Leu Ile Leu Lys Leu 245 250 255 Ile Asn Pro Glu Gly Lys Ala Tyr His Ile Ala Ala Ala Phe Pro Ser 260 265 270 Ala Cys Gly Lys Thr Asn Leu Ala Met Ile Thr Pro Thr Ile Pro Gly 275 280 285 Trp Thr Ala Gln Val Val Gly Asp Asp Ile Ala Trp Leu Lys Leu Arg 290 295 300 Glu Asp Gly Leu Tyr Ala Val Asn Pro Glu Asn Gly Phe Phe Gly Val 305 310 315 320 Ala Pro Gly Thr Asn Tyr Ala Ser Asn Pro Ile Ala Met Lys Thr Met 325 330 335 Glu Pro Gly Asn Thr Leu Phe Thr Asn Val Ala Leu Thr Asp Asp Gly 340 345 350 Asp Ile Trp Trp Glu Gly Met Asp Gly Asp Ala Pro Ala His Leu Ile 355 360 365 Asp Trp Met Gly Asn Asp Trp Thr Pro Glu Ser Asp Glu Asn Ala Ala 370 375 380 His Pro Asn Ser Arg Tyr Cys Val Ala Ile Asp Gln Ser Pro Ala Ala 385 390 395 400 Ala Pro Glu Phe Asn Asp Trp Glu Gly Val Lys Ile Asp Ala Ile Leu 405 410 415 Phe Gly Gly Arg Arg Ala Asp Thr Val Pro Leu Val Thr Gln Thr Tyr 420 425 430 Asp Trp Glu His Gly Thr Met Val Gly Ala Leu Leu Ala Ser Gly Gln 435 440 445 Thr Ala Ala Ser Ala Glu Ala Lys Val Gly Thr Leu Arg His Asp Pro 450 455 460 Met Ala Met Leu Pro Phe Ile Gly Tyr Asn Ala Gly Glu Tyr Leu Gln 465 470 475 480 Asn Trp Ile Asp Met Gly Asn Lys Gly Gly Asp Lys Met Pro Ser Ile 485 490 495 Phe Leu Val Asn Trp Phe Arg Arg Gly Glu Asp Gly Arg Phe Leu Trp 500 505 510 Pro Gly Phe Gly Asp Asn Ser Arg Val Leu Lys Trp Val Ile Asp Arg 515 520 525 Ile Glu Gly His Val Gly Ala Asp Glu Thr Val Val Gly His Thr Ala 530 535 540 Lys Ala Glu Asp Leu Asp Leu Asp Gly Leu Asp Thr Pro Ile Glu Asp 545 550 555 560 Val Lys Glu Ala Leu Thr Ala Pro Ala Glu Gln Trp Ala Asn Asp Val 565 570 575 Glu Asp Asn Ala Glu Tyr Leu Thr Phe Leu Gly Pro Arg Val Pro Ala 580 585 590 Glu Val His Ser Gln Phe Asp Ala Leu Lys Ala Arg Ile Ser Ala Ala 595 600 605 His Ala 610 

What is claimed is:
 1. An isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of a) a polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) a polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for the polypeptide mentioned and is contained on the plasmid pEK-pckA (FIG. 1) or pEK-pckB (FIG. 2), c) a polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, d) a polynucleotide which is complementary to the polynucleotides of a), b) or c) and e) a polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b), c) or d).
 2. The polynucleotide as claimed in claim 1, wherein the polynucleotide is a preferably recombinant DNA which is capable of replication.
 3. The polynucleotide as claimed in claim 1, wherein the polynucleotide is an RNA.
 4. The polynucleotide as claimed in claim 2, comprising the nucleic acid sequence as shown in SEQ ID No.
 1. 5. DNA as claimed in claim 2 which is capable of replication, comprising (i) the nucleotide sequence shown in SEQ ID no. 1, or (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and/or optionally (v) sense mutations of neutral function in (I).
 6. The polynucleotide sequence as claimed in claim 2, which codes for a polypeptide which comprises the amino acid sequence shown in SEQ ID No.
 2. 7. A vector pEK-pckA, shown in FIG.
 1. 8. A vector pEK-pckB, shown in FIG.
 2. 9. A vector pk19mobsacBΔpck, shown in FIG. 3 and deposited in the strain E. coli DH5a under number DSM
 13047. 10. Coryneform bacteria serving as a host cell, which contain one of the vectors as claimed in claims 6 to 8 or into which the Δpck deletion has been incorporated.
 11. A process for the fermentative preparation of L-amino acids, wherein bacteria in which a) the polynucleotide as claimed in claim 1 is attenuated or the activity of the polypeptide for which the polynucleotide mentioned codes is reduced. b) the desired product is concentrated in the medium or in the cells of the bacteria and c) the product is isolated are employed.
 12. The process as claimed in claim 12 wherein bacteria of the genus Corynebacterium glutamicum are employed.
 13. The process as claimed in claim 12, wherein the attenuation is achieved by integration mutagenesis with the aid of the plasmid pK19mobsacBΔpck, shown in FIG. 3 and deposited as DSM
 13047. 14. The process as claimed in 12, wherein for the preparation of L-lysine, bacteria in which optionally at the same time the dapA gene which codes for dihydrodipicolinate synthase is over-expressed, and/or at the same time a DNA fragment which imparts S-(2-aminoethyl)-cysteine resistance is amplified, are fermented.
 15. The process as claimed in claim 12, wherein for the preparation of L-threonine, bacteria in which optionally at the same time the hom gene which codes for homoserine dehydrogenase and/or hom^(dr) or hom^(FBR) alleles which code for a “feed back resistant” homoserine dehydrogenase are over-expressed are fermented. 